The goal of this proposal is to investigate the regulation of calmodulin (CaM) in nerve cells in response to neurotransmitter activation. Acute treatment with neurotransmitters, such as dopamine, alters CaM localization in many cells and chronic treatment with psychoactive drugs increases CaM content in selective areas of brain. This proposal will examine the mechanism by which neurotransmitters alter CaM localization and content and whether a Ca2+-increasing stimulus enhances binding of CaM to selective CaM-binding proteins. This problem will be studied in cultured cells since it is necessary to have a single cell type that can be readily manipulated. We found that carbachol stimulation of muscarinic receptors coupled to polyphosphoinositol (PPI) turnover elicited a robust translocation of CaM from membranes to cytosol in human neuroblastoma SK-N-SH cells. The carbachol effect was mimicked by ionomycin and phorbol esters and the extended increase in the cytosol was blocked by cycloheximide. In this proposal, the mechanism by which a receptor coupled to PPI turnover alters CaM localization and subsequent binding to CaM- binding proteins in the human neuroblastoma cell line SH-SY5Y, a clonal cell line of SK-N-SH, will be examined. There are three specific aims. 1. The molecular mechanism of carbachol-induced translocation of CaM in SH-SY5Y cells will be determined. The hypothesis will be tested that CaM is endogenously bound to neuromodulin and/or neurogranin in the cell membranes and that this binding is disrupted by Ca2+ and protein kinase C-mediated phosphorylation. Endogenous binding of CaM to neuromodulin or neurogranin will be examined using CaM-photoaffinity labels, cross- linking reagents, immunoadsorption and a site-directed antibody. 2. The consequence and selectivity of carbachol-induced changes in CaM localization will be investigated. The hypothesis will be tested that, upon dissociation, CaM will bind to selective CaM- binding proteins in the cytosol where it can alter metabolic responses and to selective proteins in the membrane cytoskeleton. Time-dependent binding of carbachol-released CaM to CaM-binding proteins will be determined by assessing the binding of a CaM photoaffinity label scrape-loaded into SH-SY5Y cells and by immunoadsorption. The subcellular localization of the CaM-binding proteins and CaM activation of enzymes will be determined. The ability of carbachol to potentiate neurotransmitter-stimulated adenylyl cyclase activity by increasing a Ca2+-dependent binding of CaM to the catalytic subunit of adenylyl cyclase will be tested in cells transfected with the clone for CaM-stimulated adenylyl cyclase. 3. The regulation of CaM content in SH-SY5Y cells by carbachol will be determined. The hypothesis that a carbachol- initiated signal induces a relatively rapid increase in CaM mRNA in these cells will be tested using a RNAse protection assay. The effect of carbachol on CaM turnover will also be assessed. These studies should increase our knowledge of how a neurotransmitter stimulus could selectively regulate Ca2+-mediated cellular events by altering the localization or content of CaM and its binding to enzymatic or structural proteins.